本文選題：Solitalea + canadensis　； 參考：《微生物學(xué)報(bào)》2017年08期

【摘要】：【目的】對(duì)細(xì)菌Solitalea canadensis中編碼β-N-乙酰氨基己糖苷酶的基因進(jìn)行克隆,通過原核表達(dá)獲得重組β-N-乙酰氨基己糖苷酶,并研究其酶學(xué)性質(zhì)�！痉椒ā恳許olitalea canadensis基因組DNA為模板,使用加尾PCR的方法克隆編碼β-N-乙酰氨基己糖苷酶的基因,構(gòu)建含有組氨酸標(biāo)簽的重組表達(dá)載體,并將重組質(zhì)粒導(dǎo)入大腸桿菌BL21(DE3)中進(jìn)行原核表達(dá)。重組蛋白經(jīng)Ni-NTA純化,以對(duì)硝基苯酚-β-乙酰氨基葡萄糖(pNP-β-Glc NAc)為底物研究其酶學(xué)性質(zhì),包括最適溫度、最適p H以及金屬離子和抑制劑的影響。【結(jié)果】從菌株Solitalea canadensis克隆得到了β-N-乙酰氨基己糖苷酶基因片段(Gene Bank:WP_014682183.1),全長(zhǎng)2586 bp,重組表達(dá)所得蛋白表觀分子量約為97 k Da,最適pH 6.0,最適溫度42°C,但不穩(wěn)定,半衰期小于5 min。該酶對(duì)十二烷基磺酸鈉(SDS)敏感,活性受Triton X-100和尿素的抑制。此外二糖分子也能不同程度地抑制該重組酶的活性,特異性抑制劑PugNAc(O-(2-Acetamido-2-deoxy-D-glucopyranosylideneamino)N-phenylcarbamate)對(duì)該酶的IC_(50)為2μmol/L。該重組酶蛋白除能水解對(duì)硝基苯酚-β-乙酰氨基葡萄糖苷和對(duì)硝基苯酚-β-乙酰氨基半乳糖(pNP-β-GalNAc)外,還能對(duì)O-鏈聚糖核心結(jié)構(gòu)Core Ⅱ末端的乙酰氨基葡萄糖進(jìn)行水解�！窘Y(jié)論】本文首次從Solitalea canadensis中克隆得到能水解末端β1-6連接的乙酰氨基葡萄糖而不能水解β1-4連接鍵的β-N-乙酰氨基己糖苷酶,并對(duì)其進(jìn)行了酶學(xué)性質(zhì)研究和底物特異性分析,為開發(fā)高效特異性強(qiáng)的糖鏈分析工具酶提供理論基礎(chǔ)。
[Abstract]:[Objective] to clone the gene of beta -N- acetaminosidase encoding beta -N- glucosidase in bacterial Solitalea canadensis and obtain recombinant beta -N- acetylglucosidase through prokaryotic expression and study its enzymatic properties. [Methods] the Solitalea canadensis genome DNA was used as a template to clone and encode beta -N- acetaminophen by adding tail PCR. The recombinant expression vector containing the histidine label was constructed, and the recombinant plasmid was introduced into the Escherichia coli BL21 (DE3) for prokaryotic expression. The recombinant protein was purified by Ni-NTA, and its enzymatic properties were studied with p-nitrophenol beta acetaminosamine (pNP- beta -Glc NAc) as the substrate, including the optimum temperature, the optimum P H and the metal ions. [results] [results] the gene fragment of beta -N- acetylhexosidase (Gene Bank:WP_014682183.1) was cloned from strain Solitalea canadensis. The total length of the gene was 2586 BP. The apparent molecular weight of the recombinant protein was about 97 K Da, the optimum pH 6, the optimum temperature 42 degree C, but unstable and the half-life less than 5 min., the enzyme was twelve alkyl. Sodium sulfonate (SDS) is sensitive and the activity is inhibited by Triton X-100 and urea. In addition, two sugar molecules can also inhibit the activity of the recombinant enzyme in varying degrees. The specific inhibitor PugNAc (O- (2-Acetamido-2-deoxy-D-glucopyranosylideneamino) N-phenylcarbamate) has a IC_ (50) of 2 micron to the enzyme and the recombinant protein can hydrolyze p-nitrophenol in addition to the enzyme. Beta acetaminoglucoside and p-nitrophenol - beta acetaminophen galactose (pNP- beta -GalNAc) can also hydrolyze acetyl glucosamine at the end of Core II at the core structure of O- chain. [Conclusion] this paper was first cloned from Solitalea canadensis to hydrolyze the acetylglucosamine linked to the terminal beta 1-6, which could not be hydrolyzed. Beta - -N- acetaminoglycosidase (beta -N-), which is a bonding bond, has been studied and analyzed by substrate specificity, which provides a theoretical basis for the development of a highly efficient and specific sugar chain analysis tool enzyme.
【作者單位】： 南京農(nóng)業(yè)大學(xué)食品科學(xué)技術(shù)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(31371739,31671854) 中央高�；究蒲袠I(yè)務(wù)費(fèi)(KYRC201209)~~
【分類號(hào)】：Q78;Q936

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